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Abstract

Group 2 innate lymphoid cells (ILC2) are a recently characterized cell population which lacks specific antigen receptors and contributes to immune responses at mucosal surfaces of lung and gut. Recently, we demonstrated that ILC2 expand in the context of chronic liver diseases in mice and contribute to pathology in an IL-33 dependent manner. Here, we describe a protocol to isolate highly purified ILC2 from mouse livers. This procedure provides effective digestion of liver tissue and limits proteolytic degradation of cell surface receptors leading to increased yields of biologically functional cells. The number of liver resident ILC2 in the steady state is low, however their number dramatically increase upon systemic or local treatment of mice with cytokines such as IL-25 and IL-33. Using minicircle-based expression constructs for these cytokines high numbers of functional ILC2 can be isolated with the protocol provided here.

Sacrifice mouse by cervical dislocation and remove the liver from a mouse aseptically under the sterile bench and transfer the organ into a well of a sterile 6 well plate filled with 5 ml KRB buffer. If co-isolation of some ILC2 cells potentially present in the vasculature should be avoided include liver perfusion e.g. via the portal vein system using standard techniques described elsewhere.

Rinse the liver with KRB buffer and transfer it into the C tube containing pre-warmed liver digesting solution.

Dissociate the tissue by applying the C tube into the gentleMACS Dissociator and running the program m_liver-01.02. Note: One mouse liver can be proceeded per C tube, the program can be run 2-3 times till the tissue is fully homogenized.

Attach the C tube containing the homogenized tissue to the MACSmix Tube Rotator and incubate the sample at 37 °C for 30 min under continuous rotation.

Apply the tube to the gentleMACS Dissociator again and run the program m_liver-02.02.

Prepare a 50 ml tube and a 100 µm cell strainer by rinsing them with PEB buffer.

Apply the homogenized tissue from C tube on the strainer on the 50 ml tube and wash the strainer with additional 10 ml PEB buffer.

Discard the cell strainer and add further 20 ml PEB buffer to the tube. Centrifuge the sample at 20 x g at 4 °C for 4 min. This step removes unwanted hepatocytes from the liver cell suspension.

Carefully remove the supernatant from the sample and transfer it into a new 50 ml tube.

This work was supported by the Collaborative Research Center 796 and the Priority program SPP1656 of the DFG (to S.W.) and the Interdisciplinary Center for Clinical Research (IZKF) of the University Medical Center Erlangen.

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